CN106434558A - Method for fish mononuclear/phagocyte separation, cultivation and functional verification - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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Abstract
The invention provides a method for fish mononuclear/phagocyte separation, cultivation and functional verification. Fish phagocytic cells are successfully obtained through optimizing cultivation parameters and conditions; functions of the fish phagocytic cells are obtained by utilizing the phagocytic cells for phagocytizing exogenous chicken erythrocyte and detecting in-vitro respiratory burst. The method provided by the invention realizes in-vitro separation, conversion, continuous cultivation and the like of the fishphagocytic cells, requires few samples, is short in operation time, simple in flow and high in accuracy, and can be widely applied in the research of fish immune-related functions on a cellular level.
Description
Technical field
The invention belongs to biological cell culture technique field, relate in particular to one kind and be used for Fish monokaryon/phagocyte
Separate, cultivate and function verification method.
Background technology
With the expansion cultivating scale in recent years, the disease problem being caused by noxious bacteria such as Vibrio anguillarums seriously hinders height
Economic worth Fish（As Cynoglossus semilaevis）The development of aquaculture industry.There is no effective immune protection technology and method at present, still
The generation to control disease by the way of chemicalses and antibiotic etc. are traditional.Peripheral blood lymphocytes（Before cytophagous
Body）, it is the important component part of Fish humoral immunization, play important during Fish specific immunity and nonspecific immunity
Effect.Studies on immune function monocytic for Fish such as Cynoglossus semilaevis still belongs to the starting stage at present, its effect unclear
Mechanism, and do not obtain the Cynoglossus semilaevis phagocyte that can continuously cultivate for cell function research so far.
Cytophagous precursor is mononuclear cell, and cell is spherical in shape, justify pyriform or irregular shape, and there is elongated pseudopodium on surface
Sample cytoplasmic process.Monocytic core is less, the kidney-shaped or shape of a hoof, often relatively side, nucleocytoplasmic ratio<1.In Giemsa dyeing, single
The nucleus of nucleuss are in latticed.Under transmission electron microscope, in its kytoplasm, there is more mitochondrion, sometimes also Golgi body, thick
Face type endoplasmic reticulum, ribosome and the senile cell being swallowed or its nucleus etc..
Mononuclear cell has stronger phagocytic activity, plays important nonspecific phagocytosis in Fish body.
It can catch and merge foreign substance and the senile cell of itself by pseudopodium sample cytoplasmic process, and can produce active deformation fortune
Dynamic.In addition, mononuclear cell is also very sensitive to environmental change, in the Fish moving in severe contamination waters, monocytic
Content is high than normal condition nearly 50 times.In Fish by parasitic infection or after artificially being injected the materials such as glue carbon granule,
The abruptly increase of amount of mononuclear cells can be caused.Therefore, by measuring the monocytic quantity of Fish, the strong of Fish can tentatively be understood
Health situation and its environmental quality in life waters.
Phagocyte after mononuclear cell and monocyte transformation plays important in opposing and defence pathogenic infection
Effect, has played huge effect in pathogen defence especially in the cell.In addition, phagocyte is also present in being swallowed
In fragment after cell phagocytosis, they also play vital effect in the natural immunity and acquired immunity.According to group
Knit different differentiation and the active phase residing for interior environment and their own, they all show different functions, form and generation
Thank to effect.Hemopoietic precursor cell in bone marrow for the cells of monocytic origin, it just migrates to each group through the blood circulation of 2-3 d
In the middle of knitting, thus being differentiated to form the phagocyte in tissue.
Acquisition can in vitro long-term surviving and can Secondary Culture cell, be by the basis of the researchs such as cellular immunology.
Common monocyte separating method includes：Plasmagel method, standard Ficoll method and Percoll-paque method.But these
Method is individually with the easy mixing suspension producing lymphocyte, granulocyte, platelet and various erythrocyte.
Content of the invention
For the deficiencies in the prior art, in order to solve the above problems, the present invention proposes a kind of monocytic for Fish
Separate, cultivate, convert and function verification method.Specific scheme is as follows：
One kind separates for Fish monokaryon/phagocyte, cultural method, and it comprises the following steps：
Step one：Monocytic separation and culture；
Step 2：Mononuclear cell induces as cytophagous In vitro culture；
Further, in step one, comprise the following steps that：
S1:Extract Fish peripheral blood, with whole blood and Hank ' s buffer 1:2.5-1:3 dilutions mix to obtain dilute blood；
S2:Take lymphocyte separation medium between dilute blood 1.5 times amount, proportion 1.05-1.09 and release blood in centrifuge tube
In；
S2:Horizontal centrifuge 1800-2100rpm is centrifuged 18-21 minute, collects mononuclearcell layer；
S4:Hank ' the s buffer adding 4-5 times of volume is washed 2 times, and 900-1100rpm centrifugation 5min collects cell precipitation；
S5:It is resuspended in DMEM complete medium, be seeded in culture bottle and be placed in 24 DEG C of incubators culture 3 h；
S6:Hank ' s buffer washes away supernatant, adds fresh culture to continue culture attached cell；
S7:Centrifugation obtains mononuclear cell layer, that is, include lymphocyte and mononuclear cell；
S8:Mononuclearcell after separation is resuspended in add and promotees adherent growth factor bFGF and contain the complete of 10-15% hyclone
In full DMEM culture medium；
S9:Wash away suspension lymphocyte after culture 2-4h, obtain adherent mononuclear cell.
Further, in step 2, comprise the following steps that：
S1:Concentrate the lymphocyte adding 10-15% in the complete DMEM culture containing mononuclear cell, 15% hyclone；
S2:Half amount exchanges fresh DMEM complete medium for every other day, cultivates 3-12 days.
A kind of monokaryon/cytophagous function verification method, it comprises the following steps：
S1:Add phorbol exters by separating the mononuclear cell obtaining（PMA）Stimulant 50ul, is incubated 15min at 23-25 DEG C；
S2:Plus Dihydrorhodamine 25ul, lucifuge incubation 5min at 24 DEG C；
S3:Washed 2 times using PBS；
S4:1500rpm is centrifuged 5min；
S5:Remove supernatant, plus 0.5mlPBS buffer re-suspended cell；
S6:Use flow cytomery.
The invention has the beneficial effects as follows：
The present invention carries out density gradient centrifugation with cell separation liquid, by revising the various parameters in experimentation and experiment bar
Part, successfully isolates and purifies Fish mononuclear cell（Phagocyte）, then pass through to optimize nutrition and condition of culture, by Cynoglossus semilaevis
Mononuclear cell is cultivated more than one week in the way of adherent growth, can be satisfied with subsequent experimental.It is thin that the present invention is successfully made phagocytosis
The respiratory burst detection of born of the same parents, is further investigation monokaryon/cytophagous biological characteristicses and immunologic function provides material.
Brief description
Fig. 1 is mononuclear cell form in the embodiment of the present invention（A：Culture 12h；B：Culture 24h；C：Culture 32h；D：Culture
38h）；
Fig. 2 is monocytic growth curve in the embodiment of the present invention;
Fig. 3 is the respiratory burst detection under PMA stimulation in the embodiment of the present invention；
Fig. 4 is Vibrio anguillarum metainfective respiratory burst detection in the embodiment of the present invention；
Fig. 5 is mononuclear cell and phagocyte in the embodiment of the present invention；
Fig. 6 is cytophagous several forms in the embodiment of the present invention；
Fig. 7 is aging phagocyte in the embodiment of the present invention；
Fig. 8 is the phagocyte of Ji's nurse Sa dyeing in the embodiment of the present invention；
Fig. 9 is the phagocyte swallowing chicken red blood cell in the embodiment of the present invention；
Figure 10 is to cultivate the 5th day phagocyte chromosome division phases in the embodiment of the present invention；
Figure 11 is phagocyte Chromosome number distribution in the embodiment of the present invention.
Specific embodiment
In order that those skilled in the art more fully understand technical scheme, with reference to the accompanying drawing of the present invention, right
Technical scheme carries out clear, complete description, and based on the embodiment in the application, those of ordinary skill in the art exist
The similar embodiment of other being obtained on the premise of not making creative work, all should belong to the scope of the application protection.
The present invention is to be completed based on the following discovery of inventor：In Fish peripheral blood cells, various cells due to
Form and the difference of weight, lead to when carrying out density gradient centrifugation：The maximum erythrocyte of proportion and granulocyte, that takes second place is single
Nucleuss (include lymphocyte and mononuclear cell), and the minimum thrombocyte of proportion can be in different levels when separating.Cause
This, by revising the density content of centrifugal rotational speed, centrifugation time, and separating liquid it is possible to utilize the side of density gradient centrifugation
Method, above-mentioned cell is carried out separating.After centrifugation, transparent plasma layer can be divided into from top to bottom, in the mononuclearcell of tunica albuginea shape
Layer, transparent separation liquid layer, granulocyte and erythrocyte proportion maximum, sink to bottom, thus reaching the purpose of crude separation；Its
Secondary, lymphocyte in tunica albuginea layer and the ability adherent when cultivating in the medium of phagocyte have differences, and therefore pass through not
Lead to the differentiation of adherent power, phagocyte can be successfully separated, the serum in cell culture medium can promote cell growth, and contributes to
Cell attachment, therefore passes through to revise the addition of serum and adds opportunity, after culture certain time, dense by revising pancreatin
Degree, to digest attached cell and can efficiently separate phagocyte and lymphocyte；And, mononuclear cell can be by phagocyte in vitro
Colony stimulating factor（M-CSF）Activation phagocytoblast, phagocyte has phagocytic activity, the feature such as respiratory burst.This is to drench
Maximum difference between bar cell and phagocyte, can identify phagocyte by this condition.
Embodiment：, the one kind introducing the present invention is used for the cytophagous culture of Fish, functional verification taking Cynoglossus semilaevis as a example
And cytophagous method for transformation.
1. the separation of Cynoglossus semilaevis peripheral blood lymphocytes and culture
Cynoglossus semilaevis are anaesthetized in frozen water, and 70% cotton ball soaked in alcohol wipes back tail vein, and anticoagulant heparin pipe extracts peripheral blood 2ml,
By whole blood and Hank ' s buffer 1 in superclean bench:2.5-1:3 dilutions mix, and take the lymph between proportion 1.05-1.09
Cell separation liquid 3ml, in 15ml centrifuge tube, adds 2ml dilute blood to be centrifuged 18-21 in horizontal centrifuge 1800-2100rpm
Minute, collect mononuclearcell layer, add Hank ' the s buffer of 4-5 times of volume to wash 2 times, 900-1100rpm centrifugation 5min receives
Collection cell precipitation, is resuspended in DMEM complete medium, is seeded in culture bottle and is placed in 24 DEG C of incubators culture 3 h, Hank ' s buffers
Liquid washes away supernatant, adds fresh culture to continue culture attached cell, half amount renews fresh complete DMEM culture medium every other day, is inverted
Basis of microscopic observation cellular morphology.
Using the difference of specific gravity of Cynoglossus semilaevis peripheral blood difference cell, centrifugation obtains mononuclear cell layer, that is, includes lymph thin
Born of the same parents and mononuclear cell, according to lymphocyte in vitro not adherent in suspended state, and the easily adherent feature of mononuclear cell, will divide
From after mononuclearcell be resuspended in add and promote adherent growth factor bFGF and contain the complete DMEM of 10-15% hyclone to cultivate
In base, wash away suspension lymphocyte after culture 2-4h, obtain adherent mononuclear cell, more than one week can be cultivated, inverted microscope
Resulted in monocyte form Fig. 1.12～16 μm of cell space diameter, cell assumes sector, the irregular form such as polygon, and kytoplasm amount is many,
There is stronger adherence quality.
2.MTT method detects monocytes in vitro growth curve
Detached mononuclear cell is inoculated in 96 well culture plates, is placed in 24 DEG C of cultures in incubator.Respectively 0h, 6h, 12h,
18h, 24h, 36h, 48h add 50ul MTT solution, and incubator continues incubation 4h.Clean supernatant, every hole adds 150ul DMSO, puts down
Plate shaking table 10min, the optical density in microplate reader detection every hole at 570nm wavelength, in triplicate.The Cynoglossus semilaevis of mtt assay detection
Monocytes in vitro growth curve is shown in Fig. 2.Cell just can be adherent well in inoculation 3h, breeds rapidly, 48h cell number after 12h
Amount is basicly stable, and cell assumes gathering growth phenomenon, form irregular shape.
3. the mensure of mononuclear cell respiratory burst
Separate the mononuclear cell obtaining and add phorbol exters（PMA）Stimulant 50ul, 23-25 DEG C of incubation 15min, plus Dihydrorhodamine
25ul, 24 DEG C of lucifuges are incubated 5min, and PBS washs 2 times, and 1500rpm is centrifuged 5min, removes supernatant, plus 0.5mlPBS delays
Rush liquid re-suspended cell, flow cytometer detects, matched group is set, matched group is added without mononuclear cell；Simultaneously directly half-and-half sliding
Tongue sole carries out Vibrio anguillarum infection：If experimental group and matched group, experimental group is with 3.18 × 105CFU/g (half lethal dose, LD50)
Cynoglossus semilaevis are carried out with lumbar injection, matched group is with reference to the PBS injection of experimental group injection and body weight corresponding dosage.After 24h respectively
Aseptic take experimental group fish and matched group fish peripheral blood, separate according to the method described above and obtain mononuclear cell, plus phorbol exters（PMA）Stimulate
50ul24 DEG C of incubation 15min of agent, plus Dihydrorhodamine 25ul, 24 DEG C of lucifuges are incubated 5min, and PBS washs 2 times,
1500rpm is centrifuged 5min, removes supernatant, plus 0.5mlPBS buffer re-suspended cell, respectively flow cytometer detection.
Flow cytomery result such as Fig. 3, the cell proportion fluorescing in normal cell controls group is 0.4%, and tests
The cell proportion that mononuclear cell under PMA stimulates in group fluoresces is 80.67%, illustrates that the mononuclear cell that separation obtains has and exhales
Inhale outburst function.The monocytic respiratory burst such as Fig. 4 obtaining, the cell fluorescing in matched group is separated after Vibrio anguillarum infection
Ratio is 4.18%, and the cell proportion fluorescing in experimental group is 83.81%, illustrates that cause of disease stimulation significantly increases mononuclear cell and exhales
Inhale outburst intensity.
4. mononuclear cell induces as cytophagous In vitro culture
Mononuclear cell and lymphocytes in vitro co-culture, and monocyte activation can be stimulated to be changed into typical phagocyte.Therefore exist
The lymphocyte adding 5-10% quantity ratio is concentrated in complete DMEM containing 15% hyclone culture, every other day half amount exchange for fresh
DMEM complete medium.After cell culture 3d, observe under inverted phase contrast microscope（As Fig. 5）.Phagocyte is adherent, and cell is stretched
Exhibition, firmly sticks, volume significantly increases, gradually becomes ellipse from irregular shape, and in fried egg shape, minority is in spindle shape.Cell
Between can have partial fusion, the visible lamellipodia of cell periphery or the two poles of the earth and projection, cell no had significant proliferation (as Fig. 6), cultivate to
13 days, phagocyte occurred aging（As Fig. 7）.
5. phagocyte dyeing is observed
Phagocyte was cultivated to 7 days, and PBS washes away the lymphocyte suspending in culture bottle, plus methanol fixes adherent gulping down
Phagocyte, Ji's nurse Sa dyeing liquor that Deca has diluted makes it cover bottom of bottle, and room temperature dyes 20min, sucks dye liquor, distilled water flushing
Attached cell, it has been observed that phagocyte cell volume is larger, form is irregular for inverted microscope.Endochylema enriches, pale pink,
Rich in granule and a little cavity.Karyon royal purple is red, is oval, kidney shape or irregular shape, has typical phagocyte shape
State feature (Fig. 8).
6. the checking of phagocyte phagocytic function
Chicken red blood cell phagocytosis test：Cultivate 5d and add 10-15% chicken erythrocyte suspension 100ul/ml in cultivating system, 24 DEG C
Incubator culture incubation 30 min to 1h, microscopy after PBS flushing.The phagocyte of phagocytosis chicken red blood cell, visible multiple in endochylema
The red born of the same parents of chicken, core is extruded (see Fig. 9).External pathogenic bacteria invades phagocyte, and microscopy observation culture medium was muddy before this, continued to be placed in 24
The culture of DEG C incubator, finds phagocyte pathogenic bacteria phagocytosis the most at last, and culture medium becomes to clarify.
7. phagocyte chromosome karyotype analysis
Carry out karyotyping when phagocyte was cultivated to the 5th day, prepare chromosome step as follows：Autumn waters -- limid eyes are added in culture bottle
Celestial element, concentration is 1ug/ml, effect 3-4h, inhales and abandons culture medium, PBS rinses, and is digested with 0.25% pancreatin and adds after mixed liquor digestion
Enter culture medium piping and druming, phagocyte is adherent relatively tight, and heavy cell is scraped using cell, then by cell suspension move into 15ml from
Heart pipe, is centrifuged 5min with 1500rpm, inhales and abandons supernatant, after the 0.0375M KCl of addition 5ml in 37 DEG C of water-baths
Hypotonic treatment 25min, is slowly added to the pre-cooling Kano fixative that 1ml newly joins, and is centrifuged min with 1000rpm, inhales and abandons
Clearly, add 2ml Kano fixative, ice bath fixing process 15min, 5min be centrifuged with 1000rpm, repetitive operation twice,
After fixing for second, how many 0.2-0.5ml Kano fixative, dropping through a cold drop method are retained according to the cell being collected by centrifugation, wait to carry
After slide is dried, dye 25min with 5%Gimsa, oil mirror microscopy after being dried.
Chromosome karyotype analysis have been carried out to the culture phagocyte of 5 days, in 100 split coil method of statistics, dyeing
Body number is 42 from 28-56, wherein 61% split coil method chromosome number（Figure 10）, 42 chromosomes are entirely
Telocentric chromosome, and wherein comprise W heterotropic chromosome.Chromosome number is in normal distribution, diploid chromosome number
Mesh frequency of occurrences highest, other aneuploid proportion very littles（Figure 11）.
Below the present invention is described in detail, the above, the only better embodiment of the present invention, when can not limit
Determine the scope of the present invention, that is, all according to the made impartial change of the application scope and modification, all should still belong in covering scope of the present invention.
Claims (4)
1. a kind of separate for Fish monokaryon/phagocyte, cultural method it is characterised in that：It comprises the following steps：
Step one：Mononuclear cell（Phagocyte precursor）Separation and culture；
Step 2：The identification of mononuclear cell body function；
Step 3：Mononuclear cell induces as phagocyte.
2. according to claim 1 a kind of separate for Fish monokaryon/phagocyte, cultural method it is characterised in that：Step
In rapid one, comprise the following steps that：
S1:Extract Fish peripheral blood, with whole blood and Hank ' s buffer 1:2.5-1:3 dilutions mix to obtain dilute blood；
S2:Take the lymphocyte separation medium between dilute blood 1.5 times amount, proportion 1.05-1.09, mixed in centrifuge tube with blood
Close；
S2:Horizontal centrifuge 1800-2100rpm is centrifuged 18-21min, collects mononuclearcell layer；
S4:Hank ' the s buffer adding 4-5 times of volume is washed 2 times, and 900-1100rpm centrifugation 5min collects cell precipitation；
S5:It is resuspended in DMEM complete medium, be seeded in culture bottle and be placed in 24 DEG C of incubators culture 3 h；
S6:Hank ' s buffer washes away supernatant, adds fresh culture to continue culture attached cell；
S7:Centrifugation obtains mononuclear cell layer, that is, include lymphocyte and mononuclear cell；
S8:Mononuclearcell after separation is resuspended in add and promotees adherent growth factor bFGF and contain the complete of 10-15% hyclone
In full DMEM culture medium；
S9:Wash away suspension lymphocyte after culture 2-4h, obtain adherent mononuclear cell.
3. according to claim 1 a kind of separate for Fish monokaryon/phagocyte, cultural method it is characterised in that：Step
In rapid three, comprise the following steps that：
S1:Concentrate the lymphocyte adding 5-10% in the complete DMEM culture containing mononuclear cell and 15% hyclone；
S2:Half amount exchanges fresh DMEM complete medium for every other day, cultivates 3-12 days.
4. a kind of monokaryon/cytophagous function verification method it is characterised in that：It comprises the following steps：
S1:If experimental group and matched group, experimental group is with 3.18 × 105CFU/g (half lethal dose, LD50) enters to Cynoglossus semilaevis
Row lumbar injection, matched group is with reference to the PBS injection of experimental group injection and body weight corresponding dosage；Aseptic respectively after 24h take experimental group
Fish and matched group fish peripheral blood, the method according to claim 2 separates and obtains mononuclear cell；
S2:Add phorbol exters by separating the mononuclear cell obtaining（PMA）Stimulant 50ul, is incubated 15min at 23-25 DEG C；
S3:Plus Dihydrorhodamine 25ul, lucifuge incubation 5min at 24 DEG C；
S4:Washed 2 times using PBS；
S5:1500rpm is centrifuged 5min；
S6:Remove supernatant, plus 0.5mlPBS buffer re-suspended cell；
S7:Use flow cytomery.
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